In vivo substrates of proteasomes are short-lived proteins regulating myriads of intracellular processes. Furthermore, aberrantly folded, damaged or malignant proteins are scavenged by proteasomes. Malfolded secretory proteins were found to be retrograde-translocated from the ER into the cytoplasm for final proteasomal degradation. Thus, proteasomes partake in endoplasmic retic-ulum-associated degradation (ERAD) as initially detected in the yeast system (24). Dysfunctions in ER-associated degradation are connected with multiple diseases, which reveal the importance of proteasome function in ERAD. In contrast to substrate breakdown, proteasomal proteolysis can also be restricted to distinct domains of a given protein, such as known for transcription factors that are released into their active form by limited proteasomal proteolysis (25).
Usually substrates are signalled for destruction by polyubiquitylation, but there is increasing evidence that proteasomes are actually able to degrade non-ubiquitylated proteins, as long as they are adequately unfolded. Beside chro-mogenic tri- and tetrapeptides commonly used in assays for proteasomal activities longer oligopeptides and small proteins (occasionally denatured) are accepted substrates of 20S proteasomes. A multitude of studies characterized the length distribution of the digestion products, which ranges between 3 and more than 20 amino acids. Preferred cleavage motifs within peptides and the impact of residues proceding and following a given cleavage site were comprehended. The data were interpreted by statistical calculations in order to allow predictions of the digestion pattern of a given protein (26). Exploiting the power of yeast genetics, wild type and mutant 20S proteasomes with inactive P1 and P2 were used to address the peptide cleavage preferences of each catalytic subunit. By soaking permeabilized yeast mutant cells with artificial peptide substrates it could be confirmed that the post-glutamyl splitting and trypsin-like activity of the proteasome is abolished upon P1 and P2 inactivation. Proteolysis of a natural protein by mutant proteasomes with inactive P1 and P2 yielded almost no digestion products with C-terminal acidic and basic amino acids, respectively (27).
Protein substrates enter the 20S proteasome by the a ring pore which in the 26S configuration is most likely gated by the adjacent ATPase ring of the 19S regulatory complex (28). Here, substrates are thought to be fed as unfolded chains from their termini into the catalytic cavity and progressively degraded. However, this mechanism cannot account for proteasome-dependent processing of transcription factor domains from inactive proproteins. Natively disordered substrates were generated and offered to latent 20S proteasomes. The disordered polypeptide chains were cut at internal peptide bonds even when they lacked accessible termini suggesting that substrates themselves are able to promote gating of the a ring pore. Thus, the endoproteolyic machinery of the 20S proteasome may provide a molecular mechanism which allows to access internal folding defects of multidomain proteins without the alliance of 19S regulatory complexes (29).
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